Plastic microfluidic separation and detection platforms

ABSTRACT

Plastic electrophoresis separation chips are provided comprising a plurality of microfluidic channels and a detection window, where the detection window comprises a thin plastic; and the detection window comprises a detection region of each microfluidic channel. Such chips can be bonded to a support provided an aperture is provided in the support to allow detection of samples in the electrophoresis chip at the thin plastic detection window. Further, methods for electrophoretically separating and detecting a plurality of samples on the plastic electrophoresis separation chip are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date, under 35 U.S.C.§119(e), of U.S. Provisional Application Ser. No. 60/921,802, filed 4Apr. 2007; and U.S. Provisional Application Ser. No. 60/964,502 filed 13Aug. 2007, and U.S. Provisional Application Ser. No. 61/028,073, filedFeb. 12, 2008, each of which is hereby incorporated by reference in itsentirety. This application also incorporates by reference, in theirentirety two US applications filed on even date herewith, AttorneyDocket No. 08-318-US, entitled “METHODS FOR RAPID MULTIPLEXEDAMPLIFICATION OF TARGET NUCLEIC ACIDS”; and Attorney Docket No.07-801-US, entitled “INTEGRATED NUCLEIC ACID ANALYSIS”.

FIELD OF THE INVENTION

This invention is in the field of nucleic acid sequencing and fragmentsizing by electrophoresis with detection by laser-induced fluorescence.The analysis is performed on plastic electrophoresis chips.

BACKGROUND OF THE INVENTION

Since the advent of DNA sequencing technologies in the 1970's (Maxam &Gilbert, 1977, Proc Natl Acad Sci USA 74: 560-564; Sanger et al., 1977,Proc Natl Acad Sci USA 74: 5463-5467), a wide range of applicationsmaking use of these technologies has developed. In parallel,increasingly sophisticated instrumentation to perform DNA sequencing hasbeen introduced. For example, in 1986, Applied Biosystems commercializedan automated DNA sequencer based on separation of DNA fragmentsgenerated by the Sanger sequencing method; DNA fragments were labeledwith a set of four fluorescent dyes and separated by capillaryelectrophoresis (Smith et al., 1986, Nature 321: 674-679). As a result,Sanger sequencing has been the most widely utilized sequencingtechnology for the last three decades.

More recently, a variety of new sequencing technologies and relatedinstrumentation have been and continue to be developed. Termed “nextgeneration” methods (reviewed in Metzker, 2005, Genome Research 15:1767-1776), these chemistries include pyrosequencing,sequencing-by-ligation, and single molecule sequencing. A major goaldriving research into next generation sequencing technologies is toperform high-throughput genomic sequencing in general, and to reduce thecost of obtaining a complete genome sequence in particular. Although thecost per base pair of next-generation technologies may be less in somecases than that of Sanger sequencing, all these methods (includingSanger) are costly and require substantial time, labor, and laboratoryequipment.

The current emphasis on obtaining very large amounts of sequence datafrom a given genome does not negate the value of obtaining relativelysmall amounts of genomic sequence quickly. For example, many commonhuman diseases can be diagnosed based on less than 1000 base pairs ofDNA sequences, orders of magnitude less than required to generate acomplete human genome. Similarly, precise determination of the sizes ofsets of less than 20 specific DNA fragments generated by short tandemrepeat analysis is sufficient to identify a given individual.

There is an unmet need for the development of instruments andtechnologies that would permit focused nucleic acid analysis, defined asthe rapid identification (by nucleic acid sequencing or fragment sizing)of a subset of a given human, animal or pathogen genome. Focused nucleicacid analysis will enable end-users to make near-real time clinical,forensic, or other decisions. Depending on the application, focusednucleic acid analysis may be performed in a variety of settings,including hospital laboratories, physician's offices, the bedside, or,in the case of forensic or environmental applications, in the field.

With respect to nucleic acid (DNA and RNA) sequencing, clinicalapplications include diagnosis of bacterial, fungal, and viral diseases(including the determination of drug resistance profiles of theorganisms), cancer (including the determination of responsiveness tochemotherapeutic regimens), and inherited and other common diseases(including the determination of responsiveness to medications). Focusednucleic acid sequencing is also well suited for pharmacogenomic analysisand certain forensic applications (including, for example, mitochondrialDNA sequencing).

With respect to nucleic acid fragment sizing, focused nucleic acidanalysis can be utilized in forensic and clinical applications. Forexample, one type of human identification is based on a short tandemrepeat (STR) analysis (Edwards et al., 1991, Am J Hum Genet49(4)746:756). In STR analysis, a series of primers are utilized toamplify certain genomic regions that contain variable numbers of certainshort tandem repeats. The sizes of the resulting bands are determined bynucleic acid fragment sizing (typically using capillaryelectrophoresis), and the size of each member of the set of STR allelesuniquely identifies an individual. STR typing has become the worldwidestandard for human forensic genetic identification and is the onlybiometric technology that allows identification of an individual as wellas genetic relatives of that individual. In clinical applications,nucleic acid fragment sizing can be used to diagnose a given disorder(e.g., by searching for a characteristic deletion or insertion, ordetermining the size of nucleotide repeat regions as in Friedreichataxia (Pandolfo, M., 2006, Methods Mol. Med 126: 197-216). Fragmentsizing is also useful for the identification of infectious agents; DNAfingerprinting can be utilized in pathogen diagnosis.

The applications of focused nucleic acid analysis are not limited tothose discussed above. Focused nucleic acid analysis can be utilized toidentify biological weapons agents in clinical and environmental samplesby both sequencing and fragment sizing. Veterinary and food testingapplications also mirror those described above. Veterinaryidentification applications such as racehorse breeding and tracking,livestock breeding, and pet identification also are within the scope ofthe uses of the disclosed invention. Research applications of focusednucleic acid analysis are numerous. In short, focused nucleic acidanalysis has the potential to dramatically transform several industries.

The existing high throughput capillary-based sequencers and the nextgeneration sequencers are not capable of performing focused nucleic acidanalysis in a timely and cost-effective fashion. The economies of scalesought by those technologies are driven by reducing the costs ofobtaining and analyzing very large amounts of sequence data. Forinstruments and systems capable of focused nucleic acid analysis to maketheir way into routine use, they should be designed to possess certain“ideal” properties and features. In particular, the instruments andsystems should generate results rapidly (ideally within minutes) toallow the generation of actionable data as quickly as possible. Theyshould be easy to operate and reagents and consumables should beinexpensive. In addition, for some applications it is useful for nucleicacid separations to be performed in disposables; this dramaticallyreduces the possibility of sample contamination. To achieve theseproperties, polymer-based biochips are better suited as separationsubstrates than other materials such as glass and silicon.

An attempt to achieve DNA fragment sizing on plastic chips was reportedby McCormick (Anal Chem 69(14):2626 1997) showing the separation ofHaeIII restriction fragments of ΦX174 RF DNA. The separations wereperformed with single samples in single lane chips, but neverthelessexhibited poor resolution separations and poor sensitivity. Furthermore,the system was only able to detect emission from a single fluorophore.Sassi (J Chromatogr A, 894(1-2):203 2000) reported the use of acrylicchips consisting of 16 fluidically isolated separation lanes for STRsizing, but this approach also showed poor resolving power and lowsensitivity. This low system sensitivity prevented the detection ofallelic ladders (internal sizing standards strictly required in forensicanalysis) when performing simultaneous 16-lane separation and detection.The use of a 2 Hz scanning rate, representing an attempt to increase thesignal to noise ratio of the system, caused degradation of bothresolving power and precision. Finally, the system was only able todetect emission from a single fluorophore. Shi (Electrophoresis24(19-20):3371 2003 and Shi, 2006, Electrophoresis 27(10):3703) reported2- and 4-color separation and detection in single sample, single laneplastic separation devices. While the 4.5 cm channel was reported toprovide single base resolution, in actuality the resolution is poor asevidenced by the appearance of alleles spaced one base pair apart (thepeak-to-valley ratio of the TH01 9.3 and 10 alleles approaches one).Devices with longer separation channels (6, 10 and 18-cm) were used inthis study to achieve higher resolution for analysis compared to the 4.5cm devices. Resolution of the 10 and 18-cm long devices were limited asthe devices delaminated when sieving matrices compositions optimized forresolution were used.

In practice, plastics have been found to present several major obstaclesfor use in biochips designed for nucleic acid sequencing and fragmentsizing. Autofluorescence of plastic materials interferes with thedetection of wavelengths in the visible range of 450 to 800 nm (Puriska,2005, Lab Chip 5(12):1348; Wabuyele, 2001 Electrophoresis22(18):3939-48; Hawkins and Yager 2003 Lab Chip, 3(4): 248-52).

These wavelengths are used in commercial kits for Sanger sequencing andSTR sizing. Furthermore, existing plastic devices have low bondingstrengths to commonly-used substrates and poor performance results withcommonly-used sieving matrices. Finally, inner surfaces of the channelinteract with sieving matrices and the DNA samples resulting in poorresolution due to electroosmotic flow and DNA-to-wall interactions (Kan,2004, Electrophoresis 25(21-22):3564).

Accordingly, there is a substantial unmet need for an inexpensive,multi-lane plastic biochip capable of performing focused nucleic acidanalysis at high resolution and with a high signal to noise ratio.

SUMMARY OF THE INVENTION

This invention provides inexpensive, multi-lane plastic biochips capableof performing focused nucleic acid analysis at high resolution and witha high signal to noise ratio and methods of using such chips.

In a first aspect, the invention provides plastic separation chips, andin particular electrophoresis chips comprising an anode portion, acathode portion, and a center portion between the anode and cathodeportions, wherein the cathode portion comprises at least one first via;the anode portion comprises at least one second via; and the centerportion comprises a plurality of microfluidic channels and a detectionwindow, each microfluidic channel having a separation region and adetection region; wherein each microfluidic channel is in fluidcommunication with at least one first via and at least one second via;wherein the plurality of microfluidic channels are in substantially thesame plane; the plurality of microfluidic channels do not intersect oneanother within the center portion; the detection window comprises a thinplastic; and the detection window comprises the detection region of eachmicrofluidic channel. The portions of the chip outside of the detectionregion can of the same thickness, or of a thickness that larger thanthat of the detection region.

In a second aspect, the invention provides devices comprising a supporthaving a top and bottom surface, comprising an anode portion, a cathodeportion, and a center portion between the anode and cathode portions,wherein the center portion comprises an aperture at the detectionwindow, the anode portion comprises the at least one anode well, and thecathode portion comprises the at least one cathode well; the apparatusfurther comprising a chip according to the first aspect, having a topand bottom surface, wherein the top surface of the chip is in contactwith the bottom surface of the support, the microfluidic channels are influid communication with the cathode and anode wells through the vias;and the chip is fixedly attached to the support.

In a third aspect, the invention provides methods forelectrophoretically separating and detecting a plurality of samplessimultaneously, comprising providing a plurality of samples into each ofa plurality of microfluidic channels on a microchip according to thefirst aspect; applying an electric potential across the plurality ofmicrofluidic channels to inject samples into the separation channel andto separate detectable species comprising each of the plurality ofanalysis samples; and detecting each of the detectable speciescomprising the plurality of separated samples at the detection window.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a microfluidic separation and detection chipaccording to the various embodiments of the invention.

FIG. 2 illustrates separate support and chip layers which can be used toconstruct a microfluidic separation and detection chip according to thevarious embodiments of the invention.

FIG. 3 illustrates separate device layers which can be used to constructa microfluidic separation and detection chip according to the variousembodiments of the invention.

FIG. 4 illustrates an expanded view of the anode section of amicrofluidic separation and detection chip according to the variousembodiments of the invention.

FIG. 5 illustrates a microfluidic separation and detection chip havinginjection channels according to the various embodiments of the invention

FIG. 6 is a schematic diagram of a microfluidic separation and detectionchip according to the various embodiments of the invention.

FIG. 7 illustrates the stack utilized for embossing.

FIG. 8 illustrates a chip support that is fabricated by CNC milling froma ⅜″ thick acrylic sheet (GE Plastic); (top) top view; bottom (sideview).

FIG. 9 is a fluorescence spectra demonstrating the low autofluorescenceof the plastic chip compared to typical glass separation chips; (a)Assembled plastic chip (Pchip2); (b) Assembled plastic chip (Pchip1);(c) plastic cover layer only; (d) glass chip, 1.4 mm thick; (e) glasschip 0.7 mm thick; (f) plastic substrate only.

FIG. 10 is an allele-called profile for the allelic ladder from a5-color labeled kit (ABI AmpFlSTR Identifiler kit); top to bottom: blue,green, yellow, red, orange detector signals.

FIG. 11 is an allele-called STR profile for 9947A human genomic DNA, topto bottom: blue, green, yellow, red, orange detector signals; fullprofile is achieved at 1.0 ng of DNA template.

FIG. 12 shows the resolution with R>0.4 for to 480 bp, demonstratingthat single base resolution to 480 bp; top to bottom: blue, green,yellow, red, orange detector signals.

FIG. 13 shows the resolution of 2 alleles (THO1 9.3 and 10) that areseparated by 1 nucleotide.

FIG. 14 is a DNA sequencing analysis of pGEM fragment; top to bottom:blue, green, yellow, and red detector signals.

FIG. 15 is a composite four base-pair graph showing a DNA sequencinganalysis of a pGEM fragment.

FIG. 16 is a breakaway schematic diagram of a chip design for directelectrokinetic sample injection showing the support (upper) and chip(bottom) layers.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides plastic separation chips that are capable ofdetecting separation of nucleic acid species differing in size by about1 basepair, and at concentration levels of at least 1.0 ng of DNAtemplate.

The lowest level of sample to be analyzed for STR analysis consists of anucleic acid template with less than 800 copies, less than 400 copies,less than 200 copies, less than 100 copies, less than 50 copies, lessthan 30 copies, less than 10 copies or 1 copy of nucleic acid templateprior to the multiplexed PCR reaction. The lowest concentration sampleto be analyzed for Sequencing consists of a nucleic acid template withless than 0.5 pmole, less than 0.1 pmole, less than 0.01 pmole as inputto the Sanger sequencing reaction.

The phrase “injection channel” as used herein, means an intersectingchannel that permits introduction of a sample into the microfluidicchannel with which it intersects. The intersecting channel can be in asingle cross-channel, a single T-junction, or an offset double-Tjunction configuration.

The phrase “fluid communication” as used herein, refers to two chambers,or other components or regions containing a fluid, connected together sothat a fluid can flow between the two chambers, components, or regions.Therefore, two chambers which are in “fluid communication” can, forexample, be connected together by a microfluidic channel between the twochambers, such that a fluid can flow freely between the two chambers.Such microfluidic channels can optionally include one or more valvestherein which can be closed or occluded, in order to block and/orotherwise control fluid communication between the chambers.

The phrase “fluorescent dye” as used herein, means the dye, uponexcitation with a light source, emits light having a wavelength of380-850 nm. Preferably, the dye emits light having a wavelength betweenabout 450-800 nm; more preferably, the dye emits light having awavelength between about 495-775 nm.

The term “autofluorescence” as used herein, means fluorescence producedby substances other than the fluorophore of interest under lightirradiation.

The phrase “essentially does not fluoresce” as used herein, means thebackground fluorescence signal (for example, between about 380-850 nm;400-800 nm; 450-800 nm; 500-800 nm, or 495-775 nm) from the referencedobject (e.g., solid or solution) when subjected to light irradiation(e.g., at one or more wavelengths between about 350-500 nm, 400-500 nm,or 450-500 nm; in particular, 488 nm; laser irradiation) has abackground level that is lower than that from conventional glassmicrofluidic devices which consist of borofloat glass of 0.7 mm thick.

The term “norbornene based polymers” as used herein means a polymerprepared from at least one monomer comprising a norbornene moiety wherethe norbornene-containing monomers are polymerized according toring-opening metathesis polymerization according to methods known tothose skilled in the art (see, for example, U.S. Pat. Nos. 4,945,135;5,198,511; 5,312,940; and 5,342,909).

The term “poly(methyl methacrylate) or “PMMA,” as used herein, means thesynthetic polymers of methyl methacrylate, including but not limited to,those sold under the tradenames Plexiglas™, Limacryl™, R-Cast™,Perspex™, Plazcryl™, Acrylex™, ACrylite™, ACrylplast™, Altuglas™,Polycast™ and Lucite™, as well as those polymers described in U.S. Pat.Nos. 5,561,208, 5,462,995, and 5,334,424, each of which are herebyincorporated by reference.

The term “polycarbonate” as used herein means a polyester of carbonicacid and glycol or a divalent phenol. Examples of such glycols ordivalent phenols are p-xylyene glycol, 2,2-bis(4-oxyphenyl)propane,bis(4-oxyphenyl)methane, 1,1-bis(4-oxyphenyl)ethane,1,1-bis(oxyphenyl)butane, 1,1-bis(oxyphenyl)cyclohexane,2,2-bis(oxyphenyl)butane, and mixtures thereof, including but notlimited to, those sold under the tradenames Calibre™, Makrolon™,Panlite™, Makroclear™, Cyrolon™, Lexan™ and Tuffak™.

As used herein the term “nucleic acid” is intended to encompass single-and double-stranded DNA and RNA, as well as any and all forms ofalternative nucleic acid containing modified bases, sugars, andbackbones. The term “nucleic acid” thus will be understood to include,but not be limited to, single- or double-stranded DNA or RNA (and formsthereof that can be partially single-stranded or partiallydouble-stranded), cDNA, aptamers, peptide nucleic acids (“PNA”), 2′-5′DNA (a synthetic material with a shortened backbone that has abase-spacing that matches the A conformation of DNA; 2′-5′ DNA will notnormally hybridize with DNA in the B form, but it will hybridize readilywith RNA), and locked nucleic acids (“LNA”). Nucleic acid analoguesinclude known analogues of natural nucleotides that have similar orimproved binding, hybridization of base-pairing properties. “Analogous”forms of purines and pyrimidines are well known in the art, and include,but are not limited to aziridinylcytosine, 4-acetylcytosine,5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, inosine, N⁶-isopentenyladenine,1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, N⁶-methyladenine, 7-methylguanine,5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,beta-D-mannosylqueosine, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acidmethylester, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid, and 2,6-diaminopurine. DNA backbone analoguesprovided by the invention include phosphodiester, phosphorothioate,phosphorodithioate, methylphosphonate, phosphoramidate, alkylphosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino),3′-N-carbamate, morpholino carbamate, and peptide nucleic acids (PNAs),methylphosphonate linkages or alternating methylphosphonate andphosphodiester linkages (Strauss-Soukup, 1997, Biochemistry36:8692-8698), and benzylphosphonate linkages, as discussed in U.S. Pat.No. 6,664,057; see also OLIGONUCLEOTIDES AND ANALOGUES, A PRACTICALAPPROACH, edited by F. Eckstein, IRL Press at Oxford University Press(1991); Antisense Strategies, Annals of the New York Academy ofSciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan,1993, J. Med. Chem. 36:1923-1937; Antisense Research and Applications(1993, CRC Press). The nucleic acids herein can be extracted from cellsor synthetically prepared according to any means known to those skilledin the art; for example, the nucleic acids can be chemically synthesizedor transcribed or reverse transcribed from cDNA or mRNA, among othersources.

The term “via” as used herein means a through-hole formed in a solidmaterial to allow fluidic connection between the top and bottom surfacesof the material.

An exemplary electrophoresis chip according to various embodiments ofthe invention is shown in FIG. 1. The chip (100) comprises an anodeportion (101), a cathode portion (102), and a center portion (103)between the anode and cathode portions. The cathode portion comprises atleast one first via (104) and the anode portion comprises at least onesecond via (105). The center portion comprises a plurality ofmicrofluidic channels (106) and a detection window (107), eachmicrofluidic channel having a separation region and a detection region;wherein each microfluidic channel is in fluid communication with atleast one first via and at least one second via. The plurality ofmicrofluidic channels are substantially in the same plane and do notintersect one another within the center portion. Each microfluidicchannel has a region in where excitation and/or detection of the samplecan take place. The area in which encompasses the excitation anddetection regions of the plurality of microfluidic channels is known asthe detection window, and this window comprises a thin plastic.

The phrase “thin plastic” as used herein, means the referenced materialcomprises a plastic having a thickness of (its smallest dimension) lessthan 1 mm, less than 750 μm, less than 650 μm, less than 500 μm, lessthan 400 μm, less than 300 μm, less than 200 μm, or less than 100 μm; orthe referenced material comprises a plastic having a thickness rangingfrom 25-2000 μm, 25-1000, 25-750 μm, 25-500 μm, 25-400 μm, 25-300 μm, or25-200 μm. Although the chip is designed to be thin in the detectionwindow, portions of the chip outside of the detection region can be ofthe same thickness, or of a thickness that is larger than that of thedetection region.

The chip of FIG. 1 is shown for the sake of illustration as having fourmicrofluidic channels, however such disclosure is not intended to belimiting, rather, one skilled in the art will readily recognize that thechip can contain alternate numbers of microfluidic channels (infra)including chips with one channel and chips with two or more channels.The term “plurality” as used herein, means two or more, four or more,eight or more, 16 or more, 32 or more, 48 or more, 64 or more, 96 ormore, 128 or more, 256 or more, 384 or more, 512 or more, or 1024 ormore; or 2-4, 2-8, 2-16, 2-32, 2-48, 2-64, 2-96, 2-128, 2-384, 2-512,2-1024 microfluidic channels.

The chip (250) comprises of a substrate layer (360) and a cover layer(370) as shown in FIG. 3. A plurality of grooves (361) are patternedinto the substrate layer. A series of vias (i.e., through holes) (371,372) are formed in the cover layer to provide fluidic access to themicrofluidic channels, and can be located at the ends of themicrofluidic channels in the anode and cathode portions of the chip.Alternatively, vias can be formed in the substrate layer instead of thecover layers to achieve the same functionality. The top surface of thesubstrate layer is bonded with the bottom surface of the cover layer toform the microfluidic channels. Techniques for fabricating polymer-basedmicrofluidic systems, reviewed extensively by Becker and Gartner(Becker, 2000, Electrophoresis 21: 12-26 and Becker, 2008,Electrophoresis 390(1):89), which are hereby incorporated by referencein its entirety. Any number of these processes can be used to fabricatethe plastic separation chip described herein.

In particular, the present plastic separation chips can be prepared byhot embossing of thin thermoplastic films with a master die of thenegative of the structure to be produced. The master die can be preparedby using electroforming to replicate the device prepared in a solidsubstrate. The solid substrate can be glass sheets that are patterned bystandard photolithographic and chemical etching methods known to thoseskilled in the art. The substrate and cover layers are diffusion bondedby the application of heat and pressure.

The substrate and cover layers of the chip can be constructed from avariety of plastic substrates including, but not limited to,polyethylene, poly(acrylates) (e.g., poly(methyl methacrylate)),poly(carbonate)s, and unsaturated, partially unsaturated or saturatedcyclic olefin polymers (COP), or an unsaturated, partially unsaturated,or saturated cyclic olefin copolymers (COC) (e.g., ZEONOR™, ZEONEX™ orTOPAS™). In particular, COP and COC are advantageous for the presentchip applications as they optically exhibit inherently lowerautofluorescence in the visible wavelength range compared with otherpolymers.

The thickness of plastic substrate and cover layers utilized in thepresent process is kept thin to minimize autofluorescence from the chip.The plastic substrate and cover layers can each, independently, have athickness of less than 2 mm, less than 1 mm, less than 750 μm, less than650 μm, less than 500 μm, less than 400 μm, less than 300 μm, less than200 μm, or less than 100 μm; or plastic substrate and cover layers caneach, independently, comprise a plastic having a thickness ranging from25-2000 μm, 25-1000, 25-750 μm, 25-650 mm, 25-500 μm, 25-400 μm, 25-300μm, 25-200 μm, or 25-100 μm.

In one embodiment, as exemplified in FIG. 2, the chip (250) is attachedto a support (201) having a top and bottom surface, comprising an anodeportion (202), a cathode portion (203), and a center portion (204)between the anode and cathode portions, wherein the center portioncomprises a detection window (205), the anode portion comprises at leastone anode well (206), and the cathode portion comprises at least onecathode well (207). The top surface of the chip, with the via holes up,is in contact with the bottom surface of the support, and the chip isfixedly attached to the support. The chip can be attached to the supportaccording to methods known to those skilled in the art, for example,diffusion bonding, solvent bonding or adhesive bonding.

The support layer can be constructed from a variety of plasticsubstrates including, but not limited to, polyethylene, poly(acrylates)(e.g., poly(methyl methacrylate)), poly(carbonate)s, and unsaturated,partially unsaturated or saturated cyclic olefin polymers (COP), or anunsaturated, partially unsaturated, or saturated cyclic olefincopolymers (COC) (e.g., ZEONOR™, ZEONEX™ or TOPAS™). The thickness of aplastic support layers utilized in the present process is sufficientlythick in order to provide structural rigidity and to allow forsufficient volume of sample and buffers in the reservoirs. The thicknessof the plastic support will range from 100-15,000 μm.

Alternatively, the chip can be fabricated by patterning the grooves onthe solid support to form both the chip substrate and support structurestogether. A cover layer can be bonded to the support to complete thestructure. In this configuration, the thickness of a detection window ofthe support and chip coincident with the detection portion of themicrofluidic channels is kept thin to minimize autofluorescence. Thethickness of this portion of the chip is less than 1000 μm, less than750 μm, less than 500 μm or less than 250 μm; or ranging from 25-1000μm, 25-750 μm, or 25-500 μm.

Each of the plurality of microfluidic channels can have a depth of atleast 10 μm, 50 μm, 100 μm, 200 μm, 500 μm or 1 mm; or have a depthranging from 1-1000 μm, 10-100 μm, 10-50, or 25-50 μm. The plurality ofmicrofluidic channels can have a width of at least 25 μm, 50 μm, 100 μm,200 μm, 500 μm or 1 mm; or have a width ranging from 25-1000 μm, 25-200μm, or 50-200 μm. The microchannel cross-section of each channel canhave a substantially square, rectangular, circular, semicircular,elliptical, triangular or trapezoidal cross-section. One skilled in theart will recognize that the microfluidic channels may or may not beuniform in depth, width and cross-section.

Each of the plurality of microfluidic channels (106) comprises aseparation region (108) and a detection region (109). The separationregion typically has channels with separation length of about 2-50 cm,10-50 cm, 2-25 cm, 10-25 cm. The separation length is defined as theportion of the channel between the point of sample injection and thepoint of sample detection. The separation length is typically less thanthe total length of the separation channel which spans between thecathode and the anode reservoirs.

Simultaneous analysis of a plurality of samples can be performed byinjecting and stacking each of the samples in a separate separationchannel into any of the separation chips described herein. Theapplication of an electric field along the separation channel causes thesamples to migrate along the channel from the cathode portion toward theanode portion or the anode portion to the cathode portion of theseparation channel, depending, for example, on the charges present onthe surfaces of the channel (infra), as will be familiar to thoseskilled in the art. Migration of the sample through a sieving matrixseparates species on the basis of size.

As the separated samples pass through the detection window dye labelsattached to each species within the sample can be excited and theresulting fluorescence can be detected. The detection window typicallyoverlaps the detection region of each of the plurality of microchannelat the termini of the separation region of each of the channels.Typically, the detection region for each of the plurality ofmicrofluidic channels are in substantially the same location along thechannels, such that the detection window can be in a single location inthe center portion of the support.

An injector for simultaneously injecting a plurality samples into theplurality of sample or buffer wells is advantageously provided with thechip to enable simultaneous multiple sample separation and detection.Such injectors provide, for example, one sample of the plurality ofsamples to one microfluidic channel of the plurality of microfluidicchannels. Injectors can introduce the samples to the channels accordingto any methods known to those skilled in the art, for example, byelectrophoretic transport, pneumatic actuation or liquid actuationthrough a needle or tube or channel that connects the sample to theseparation channel

In certain embodiments, samples can be loaded into the chip through thecathode reservoirs of the chip. An injection volume of each sample canbe introduced through one of the cathode wells according to methodsknown to those skilled in the art. For example, the sample can beinjected via appropriate biasing of the separation channel and/or across-channel of the separation channel and the sample and waste wellssuch that a portion of the sample (i.e., the injection volume) in thesample well is provided to the separation channel. Following sampleinjection, additional buffer solution is introduced into each cathodewell; sufficient volume can be provided to dilute any remaining samplein the well. For example, a volume of buffer is introduced into thecathode wells that is about at least 5, 10, 25, 50, or 100 times theinjection volume of the sample. Alternatively, a volume of buffer isintroduced into the cathode wells that ranges from about 5-100 times,5-50 times, or 10-50 times the injection volume of the sample.

In other embodiments, each of the plurality of microfluidic channelsfurther comprises an injection channel for introducing samples. Forexample, reference is made to FIG. 4; shown therein is an expanded viewof a chip (400) showing the cathode portion (401) and adjoining sectionof the center portion (403). The cathode portion comprises at least onesecond via (405) and the center portion comprises a plurality ofmicrofluidic channels (406). Each microfluidic channel furthercomprises, within the cathode portion of the chip, an injection channel(408) comprising a sample (409) and waste (410) well for eachmicrofluidic channel.

The injection channel can be in a single cross-channel (as illustratedin FIG. 4), a single T-junction, or an offset double-T junctionconfiguration. In some embodiments, the injection channel is an offsetdouble-T junction configuration that minimizes the injection volume ofsample, thereby improving separation resolution. Injection of a samplefrom the injection channel to the microfluidic channel can beaccomplished according to methods known to those skilled in the art,including electrophoretic injection through application of theappropriate potentials at the sample, waste, anode and cathode wells.

An alternative embodiment of the microfluidic separation and detectionchip is illustrated in FIG. 5. The chip (500) comprises an anode portion(501), a cathode portion (502), and a center portion (503). The cathodeportion comprises one first via (504) for each microfluidic channel(506) and the anode portion comprises at least one second via (505) foreach microfluidic channel (506). The center portion comprises aplurality of microfluidic channels (506) and a detection window (507),each microfluidic channel having a separation region and a detectionregion; wherein each microfluidic channel is in fluid communication withone first via and one second via. The plurality of microfluidic channelsare essentially in the same plane and do not intersect one anotherwithin the center portion. The detection window comprises a thin plasticand overlaps the detection region of each microfluidic channel.

In this instance, the injection channels are omitted in favor of ananode (second) and cathode (first) via for each microfluidic channel. Aninjection volume of each sample is introduced through one of the cathodevia according to methods known to those skilled in the art (supra).Following sample injection, additional buffer solution is introducedinto each cathode buffer well; sufficient volume is advantageouslyprovided to dilute any remaining sample in the well, thereby mediatingany background signal introduced from prolonged sample injection andimproving the signal-to-noise ratio observed at the detection window.For example, a volume of buffer is introduced into the cathode wellsthat is about at least 5, 10, 25, 50, or 100 times the injection volumeof the sample. Alternatively, a volume of buffer is introduced into theanode buffer wells that ranges from about 5-100 times, 5-50 times, or10-50 times the injection volume of the sample.

Electrophoretic separation of the samples within the microfluidicchannels is provided by the application of a potential difference acrossthe microchannels on the microchip. A high voltage can be applied acrossthe ends of the microchannels, typically by placing a cathode and anodein the cathode well and the anode well, respectively, establishing anelectric field along the separation portion of the microfluidic channel,and moving the sample (e.g., nucleic acid) from the cathode end throughthe separation portion to the detection portion, and ultimately, to theanode. The electric field required for efficient separation often rangefrom 50 V/cm to 600 V/cm. The voltage from the power supply is appliedthrough electrodes and a buffer is used in the anode and cathodereservoir to provide electrical contact between the electrode and thesieving polymer.

High voltages required for sample separation are applied to theseparation channel with electrodes that are in contact with the bufferthat is in the cathode and anode wells. Due to the high voltages presentat the electrodes that are in contact with the buffer, the buffer watermolecules hydrolyze resulting in the formation of OH⁻, H⁺, and H₂ gas.This formation results in a change in the pH of the buffer with time,and formation of bubbles within the buffer. The pH change of the buffercan be attenuated through sufficient use of a buffer solution in theanode and cathode reservoirs (e.g., 1×TTE; Amresco) to provide contactbetween the electrode and the sieving matrix. The bubbles formed withinthe buffer have a tendency to migrate into the sieving matrix blockingthe channel, resulting in poor separation of nucleic acids.

Bubbles that form at the electrode can be prevented from migrating intothe channel by using one or a combination of the following methods.First, the electrode within the reservoir can be raised to move thesource of bubble generation (electrode) away from the access holes inthe channels. Secondly, a glass frit, polymer frit, or polymer membraneor polymer filter can be inserted between the cathode access hole andthe end of the electrode. In particular, a polymer frit (e.g.,polyetheretherketone, PEEK) can be inserted between the cathode accesshole and the end of the electrode.

The frit, membrane, or filter is selected to be non-conducting and havea pore size that prevents bubbles formed at the electrode from passingthrough the pores. As a result of insertion of the frit, polymermembrane, or filter between the electrode and the sieving matrix,bubbles formed from the electrolysis process are prevented from enteringthe channels. This implementation can reduce and/or eliminate failuresresulting from bubble blockage in the channels.

Separation devices for simultaneous analysis consisting of a pluralityof samples are electrically connected so that a common power supply canbe used to bias the plurality of channels simultaneously. Furthermore,physical constraints of the chip and instrument will usually not allowall the channels to have an identical physical layout with respect tolength, depth and width.

To achieve substantially identical electrophoretic injection andseparation conditions for each of the plurality of microfluidicchannels, each channel segment of the individual devices should haveessentially identical resistances and hence electric fields. Asubstantially identical electric field, that is wherein the electricfields across each of the plurality of microfluidic channels does notdiffer by more than about +/−5%, can be established by simultaneouslyadjusting both the length, width and depths of each of the plurality ofmicrofluidic channels to adjust the resistance of each segment of thechannels. The resistance, R, of each segment can be described by thefollowing relationship:

$R = {\rho \frac{l}{A}}$

where ρ is the resistivity, l is the length and A is the cross-sectionalarea of the channel.

Surface charges resident on the wall of the channels of the separationchip can result in electroosmosis and sample-to-wall interactions. Theseeffects can be can minimized by applying a surface coating to the innerwalls of the microfluidic channels. Such surface coatings andmodifications can be accomplished through methods known to those skilledin the art (for example, Ludwig and Belder, 2003 Electrophoresis24(15):2481-6).

A large number of candidates for surface modification are availableincluding hydroxypropylmethylcellulose (HPMC), poly(ethylene oxide)(PEO), poly(vinyl alcohol) (PVA), polydimethyl acrylamide (PDMA),poly(vinylpyrrolidinone), dimethylacrylamide (DEA), diethylacrylamide(DEA), poly(diethylacrylamide) (PDEA), and mixtures thereof, such asPDMA:PDEA.

Additionally, for use in electrophoretic applications, each of theplurality of microfluidic channels is advantageously filled with asieving matrix. Such sieving matrices can comprise, in non-limitingexample, a linear polyacrylamide (PAA), polydimethylacrylamide (PDMA),polydiethylacrylamide (PDEA), polyvinylpyrrolydinone (PVP), andcombinations thereof, including for example, PVP:PAA, PDMA:PAA,PDEA:PAA, PDEA:PDMA:PAA. In certain embodiments, the sieving matrixcomprises 0.1-50 wt. % polyacrylamide. A number of these sievingmatrices also possess dynamic self-coating capability. As practicedusing these embodiments of the electrophoretic separation chips of theinvention, nucleic acids move electrophoretically through a sievingmatrix from the anode to the cathode end and are size-separated therein.As set forth above, the inner walls of the channels can be coated tominimize the influence of electroosmosis and nucleic acid-to-wallinteractions.

Resolution, specifically herein electrophoretic resolution, is theability to unambiguously discriminate two peaks separated in time (or bybase size). The resolution (R) is defined by the following equation

$R = {\left( {2\; \ln \; 2} \right)^{\frac{1}{2}}\frac{t_{2} - t_{1}}{\Delta \; {b\left( {{hw}_{1} + {hw}_{2}} \right)}}}$

where t is the migration time of the nth peak, hw is the full width andhalf-maximum of the nth peak, and Δb is the base number differencebetween the two peaks. Single base pair resolution is defined at thepoint where R is greater than 0.4. Visually, two peaks aredistinguishable from each other when the peak to valley ratio is greaterthan 0.7. Both R and peak-to-valley requirements must be met in order tohave high resolution, and resolution can also be considered to becharacteristic of a range of fragment sizes. The range of fragment sizesfor alleles in STR analysis range from 90 to 400 bp and single basepairresolution across this range of fragment size is required for STRanalysis. Fragment sizes for sequencing analysis range up to 1200 bp.The ability to achieve long read lengths and data throughput per laneis, in part, determined by the range over which the chip is able togenerate single base-pair resolution.

The limit-of-detection for an optical detection system is defined by thesignal to noise ratio (SNR). This ratio is defined as the ratio of asignal power to the noise power (standard deviation of noise power)corrupting the signal. A high SNR indicates a higher certainty that asignal is present. A signal-to-noise ratio of 3 is generally defined asthat which is acceptable for confidently identifying the presence of asignal (Gilder, 2007, J Forensic Sci. 52(1): 97).

When analyzing and detecting a plurality of nucleic acid species in anucleic acid sample, autofluorescence from the plastic in the detectionwindow of the chip strongly contributes to the fluorescence background.An advantageous characteristic of the electrophoretic separation chipsof the invention is that a thin detection window is used to minimize thebackground fluorescence from the plastic. This background level iscompared to Borofloat®, which is a commonly-used substrate forfabricating microfluidic separation chips. With the use of a thinplastic window, a minimum of 1000 copies, 300 copies, 100 copies, 30copies, 10 copies, 1 copy of template nucleic acid in the PCR processthat generates fluorescently-labeled fragments for analysis can bedetected. Also a minimum of 0.5 pmoles, 0.1 pmoles, 0.01 pmoles, or0.001 pmoles of nucleic acid template for the sequencing reaction can bedetected.

Separation and Detection Chip Applications

Applications of the various aspects of the invention extend broadly forboth nucleic acid identification and sequencing. Examples of uses inhuman identification include criminal forensics and homeland security,for example identification at military checkpoints, borders and ports,airports and mass disaster sites. Veterinary identification applicationsincluding racehorse breeding and tracking, livestock breeding and petidentification also are within the scope of the uses of the disclosedelectrophoretic chips.

Moreover, the instruments of this invention can be ruggedized, andthereby operated in the field where results can be used in real-time. Assuch, the instruments can be used at military checkpoints, borders andports, airports, and mass casualty sites.

Applications of the technology to nucleic acid sequencing can be dividedinto four areas: human clinical diagnostics, including, for example,bacterial infections and antibody sensitivities, viral infections(identification and drug resistance profiling), genetic diseases,complex disorders (asthma, heart disease, diabetes) andpharmacogenomics; veterinary clinical diagnostics; research sequencing,including re-sequencing and finishing; biological weapons agentidentification, including, for example, B. anthracis and Ebola virusdetection; and food safety. Some examples follow.

A patient with HIV needs drug resistance testing. Today, it can takeweeks to establish resistance. A drug resistant strain can take holdduring that time. There is an unmet need for an instrument and systemthat can provide the answer within 1-2 hours, while the patient waits inthe physician's office. Use of an electrophoretic separation chipaccording to the invention permits frequent drug-resistance monitoring,more clinically- and cost effective usage of anti-viral agents andbetter patient outcomes.

A patient with bacteremia is in shock. Today, it can take days todetermine whether the causative agent is resistant to antibiotics andthe identities thereof. In the interim, the patient must be treated withbroad-spectrum antibiotics, which can cause serious side-effects to thepatient and contributes to the increase in antibacterial resistanceprevalent today. In addition, such treatments may be sub-optimal. Use ofan electrophoretic separation chip according to the invention permitsidentification of the antibiotic resistance profile of the pathogen in1-2 hours, leading to more effective, targeted treatment, reduction inantibiotic toxicities, and better patient outcomes. The benefits to thepatient and to public health are complimentary.

A patient with cancer is undergoing surgery. Today, a tumor sample istaken to pathology while the patient is on the operating table. Based onthe results of the simple histopathology strains, a decision is madeconcerning how aggressive the surgeon should be. Use of anelectrophoretic separation chip according to the invention could replacehistopathology with a definitive nucleic acid diagnosis of the cancer inless than an hour, allowing a better-informed surgical decision to bemade.

The Examples which follow are illustrative of specific embodiments ofthe invention, and various uses thereof. They set forth for explanatorypurposes only, and are not to be taken as limiting the invention.

EXAMPLES Example 1 Chip Design and Electrophoresis Example 1A ChipDesign

A schematic diagram of a particular embodiment of the devices of theinvention is illustrated in FIG. 6. This microfluidic device consistedof 16 microchannels, each with a double-T cross injector. Thecross-sectional dimension of the channel (90 μm wide and 40 μm deep) andlength of the channel between the anode and the cross-injector (25 cm)was equal for all channels. The separation lengths (distance between theintersection and the excitation/detection window) for each of thechannels range from 16 to 20 cm long. The cross-sectional area of thechannels between the cathode well and the injector was adjusted suchthat all the resistances and hence electric fields between the cathodeand the intersection are essentially equal under bias. This ensured thatthe electric fields experienced by the samples were identical regardlessof the separation channel into which a sample was loaded. Theintersection voltages for all channels were essentially identical. Thesample inlet and sample waste arms for sample injection were both 2.5 mmlong. The offset between both channels was 500 μm.

Example 1B Chip and Support Fabrication

The chip was patterned by hot-embossing, drilling to form access holes,and diffusion bonding to seal the channels. The master was fabricated inglass by photolithography, using a chemical wet etching process. Thisglass master was then used to fabricate a nickel-cobalt embossing toolby electroforming to generate a negative replicate of the glass master.Sheets of Zenor™-1420R film (5″×2″ in size and 188 μm thick) were usedas the substrate material. On these sheets, cathode, anode, sample andwaste access holes were formed by drilling. This was followed by hotembossing the chip design features on the embossing tool into thesubstrate. Embossing was accomplished by placing the stack asillustrated in FIG. 7 in a heated hydraulic press for 15 minutes at 135°C. and 1250 psi of compressive pressure. The stack was held under 1250psi of compressive pressure and allowed to cool to 38° C. prior torelease. Fabrication of this chip with thin thermoplastic polymerscontaining norbornene monomers resulted in a low background fluorescenceat excitation and detection window. Achieving high bond strengthdiffusion bonding allowed the use of high viscosity sieving matrices.

Diffusion bonding of the substrate was achieved by the aligning a sheetof Zenor™-1420R film (5″×2″ in size and 188 μm thick) over the substrateand subjecting this stack to heat and pressure. No adhesive was appliedbetween the sheets of film; bonding was accomplished entirely by heatand pressure. The final thickness of the chip was approximately 376 μm.Separation chips fabricated by this method were tested and demonstratedto be capable of withstanding at least 830 psi of pressure beforefailure.

FIG. 8 illustrates a chip support that was fabricated by CNC millingfrom a ⅜″ thick acrylic sheet (GE Plastic). The chip support consistedof three main sections: the cathode board, the center part and the anodeboard. The cathode board contained the cathode well, sample and wastewells, and alignment holes. The anode board contained the anode well andalignment holes. Both the cathode board and the anode board were ⅜″thick to provide enough sample volume for sample injection and buffervolume for electrophoresis. The center portion was 0.04″ thick and hadan opening as the “detection window” for laser induced fluorescencedetection in the microchannels. With this configuration,autofluorescence from the separation chip becomes dominated by theapproximately 376 μm thick substrate. The separation chip was attachedto the chip support with double-sided pressure sensitive adhesive. Theadhesive was selected to be inert to the separation buffers and sievingmatrices. The support and separation chip were attached with pressuresensitive epoxy. The thickness of the plastic in the excitation anddetection area was minimized by fabricating a cut-out on the carrier inthis region.

The optical emission spectrum of Zenor™-1420R has the Raman emissionpeak at 570 nm which has limited fluorescence detection with fluorescentdyes. FIG. 9 demonstrates low autofluorescence of the plastic chip(PChip1 and PChp2) compared with typical glass separation chips (Glass1.4 mm and Glass 0.7 mm). The low autofluorescence of the plastic chipwas achieved by selecting a COP polymer and minimizing the thickness ofthe device in the detection area and by fabricating the device with thinfilms.

Example 1C Surface Modification and Sieving Matrix

Surface modification was accomplished by initially pre-treatingmicrochannel surfaces with de-ionized water, followed by 1 M NaOH. Anitrogen flush was applied to remove fluids from the channels. Treatmentof the surface was followed by flowing 0.1% (w/v)hydroxypropylmethylcellulose (HPMC) solution through the channelsfollowed by incubation overnight at room temperature. High puritynitrogen was used to flush through the channels to remove fluids insidethe channel.

The sieving matrix used for these experiments was 4% linearpolyacrylamide (LPA) in 7M Urea and 1×TTE (Amresco) buffer

Example 1D Electrophoresis STR Sizing

Electrophoretic separation and analysis of nucleic acid analysis wasperformed on the Genebench-FX™ Series 100 (Network Biosystems, Inc.,Woburn, Mass.). This instrument was configured to accept the plasticseparation chip and chip support to allow for good optical, electricaland thermal coupling between chip and instrument. The temperature of thechamber was maintained at 50° C. throughout the operation.

For DNA sizing experiments human genomic DNA was amplified with the ABIAmpFISTR kit (Applied Biosystems Inc., Foster City, Calif.). PCR product(2.7 μL) was mixed with 0.3 μL of sizing standard and 10 μL offormamide, and loaded into the sample wells for analysis. The assayconsisted of pre-electrophoresis performed at 156 V/cm for 6 minutesprior to sample introduction by applying a potential difference of 3900V at the anode well and grounding the cathode well. DNA samples wereintroduced by applying an electric field of 350 V/cm for 18 seconds,followed by a dual load of 1.2 minute by applying an electric field of350 V/cm across the sample and waste wells and simultaneously applyingan electric field of 15.6 V/cm across the cathode and anode wells. Aftersample injection, electrophoretic DNA separation was performed byapplying an electric field of 156 V/cm across the cathode and anode wellwhile maintaining a pullback voltage of 800 V for 40 minutes.

For DNA sequencing experiments, M13 plasmid was cycle-sequenced with theGE Amersham DYEnamic™ ET dye terminator cycle sequencing kit (GEHealthcare), ethanol precipitated and resuspended in 10 μL deionizedwater. The separation assay consisted of a pre-electrophoresis performedat 156 V/cm for 6 minutes prior to sample introduction by applying apotential difference of 3900 V at the anode well and grounding thecathode well. DNA sample was introduced by applying an electric field of350 V/cm for 60 seconds. After sample injection, electrophoretic DNAseparation was performed by applying an electric field of 156 V/cmacross the cathode and anode well while maintaining a pullback voltageof 400 V for 60 minutes. DNA separation resolution was calculated byextracting the peak information (peak spacing and peak width) fromPeakfit®.

Successful separation was achieved simultaneously in 16 lanes in theplastic chip. FIG. 10 shows allele called profile for the allelic ladderfrom a 5-color labeled kit (ABI AmpFlSTR Identifiler kit). These resultsdemonstrated that devices of the invention were able to separate with 5colors in a plastic chip and clearly resolve alleles including ones thatare spaced by a distance equivalent to only a single base pair (THO 1,allele 9.3 and 10). FIG. 11 shows an allele called STR profile for 9947Ahuman genomic DNA, showing that a full profile was achieved at 1.0 ng ofDNA template. FIG. 12 shows the resolution with R>0.4 for up to 480 bp,demonstrating single-base resolution up to 480 bp. FIG. 13 illustratesthis resolution by showing 2 alleles that are spaced by 1 nucleotide canbe clearly resolved with no ambiguity. FIGS. 14 and 15 shows a DNAsequencing profile demonstrating single basepair resolution.

Example 2 Electrokinetic Injection Plastic Chip Example 2A Chip Design

Another configuration of the electrophoretic separation chips of theinvention uses a single channel for separation. Each sample isintroduced into a separation channel by electrokinetic sample injection.This alternative approach allows for the use of small sample volumes anda significant simplification in the separation process. A schematicdiagram of chip design for electrokinetic sample injection is shown in abreakaway view in FIG. 16, showing the support and separation chipsections. The device consists of 16 microchannels that are effectively20 cm in separation length. Each channel has an access hole at each end.The channels are 90 μm wide and 40 μm deep.

Example 2B Device Fabrication

The device of FIG. 16 is fabricated following the procedure described inthe section above. In summary, access holes (1 mm in diameter) areformed in a COP film (Zeonor™-1420R) with a thickness of 188 μm. Channelpatterns (width 90 μm and depth 40 μm) are then formed by hot embossing.A cover of COP (Zeonor™-1420R) is diffusion bonded to the substrate toseal the channels.

Example 2C Electrophoresis

The device is prepared for separation by applying a surface modificationto the channels as described in the section above. This is followed byfilling the channels with a sieving matrix. Samples are loaded into thesample/cathode reservoir. An injection field is applied through theelectrodes to the sample to inject negatively charged DNA into theseparation channel. Following injection of DNA into the channels, buffer(1×TTE; Ameresco) is added to the sample/cathode reservoir at a volume10 times the volume of the sample. An electric field is applied acrossthe cathode and anode to separate the DNA from the injection plug downthe separation channel. The addition serves to dilute the sample that isin the sample/cathode and there is no need to remove the sample prior toloading the buffer. Separation and detection is performed on aGenebench-FX™ Series 100 instrument, and data analysis is performed withthe software described in the previous examples.

Example 3 DNA Sequencing

For DNA sequencing analysis, DNA template is amplified in a reaction mixconsisting of PCR enzyme SpeedSTAR HS (Takara, Madison, Wis.) (U/μL):0.025, Fast Buffer 1: 1×, dNTPs: 0.25 mM, Primer (forward): 250n M, andPrimer (reverse): 250 nM. A desired level of template DNA is added tothe mix. DI water or TE buffer (Tris 10 mM or EDTA 0.1 mM) is added tothe reaction mix to a total volume of 10 μL. Thermal cycling of the PCRreaction mix, following manufacturer's recommended protocols, consistsof hot start activation of 60 seconds at 95° C., 30 cycles ofdenaturation, anneal and extension (5 seconds at 98° C., 10-15 secondsat 55° C. and 5-10 seconds/kbp at 72° C.) and a final extension of 60seconds at 72° C.

The entire PCR product is cleaned up by using a 30K MWCO UF filter(Pall, East Hills, N.Y.), following manufacturers protocol. The cleanedup product, with consisting of DNA in DI water, is either diluted orapplied in its entirety as template for the sequencing reaction.

Cycle sequencing of PCR template, was performed using the DYEnamic™ ETTerminator Cycle Sequencing Kit (GE Amersham Biosciences) at halfstrength reaction with the following reaction mix. Sequencing Premix: 4μL, Dilution Buffer: 4 μL, Primer (101M): 5 pmol. DNA template was addedto the sequencing reaction mix. DI water was added to the reaction mixto a total volume of 20 μL. Following manufacturer's recommended cyclingprotocols the cycling condition used consists of thirty cycles of (20seconds at 95° C., 15 seconds at 50° C., 60 seconds at 60° C.).

The sequencing reaction mix is cleaned up by ethanol precipitation. Theprecipitated product is resuspended in 13 μL of DI water and used assample for separation and detection.

For STR analysis, amplification is carried out in 10 μL reactions withthe following reaction mix consisting of: PCR enzyme SpeedSTAR HS(Takara, Madison, Wis.) (U/μL): 0.0315, Fast Buffer 1: 1×, Primer set: 2μl, Fast Buffer 1: 1×, dNTPs: 200 μM, Primer (forward/Reverse): 2 μLfrom AmpFlSTR Profiler™, COFiler™ or Identifiler™ (Applied Biosystems,Foster City, Calif.).

The cycling protocol follows enzyme manufacturers conditions consistingof a hot start activation of 60 seconds at 95° C. followed by 28 cyclesof denaturation, anneal and extension (4 at 98° C., 15 s at 59° C., 5 sat 72° C.) and a final extension of 60 seconds at 72° C. PCR product isused as sample for separation and detection. Alternatively, the PCRproduct can also be purified and used as sample for separation anddetection.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

1. An electrophoresis separation chip comprising an anode portion, acathode portion, and a center portion between the anode and cathodeportions, wherein the cathode portion comprises at least one first via;the anode portion comprises at least one second via; and the centerportion comprises a one or plurality of microfluidic channels and adetection window, each microfluidic channel having a separation regionand a detection region; wherein each microfluidic channel is in fluidcommunication with at least one first via and at least one second via;the plurality of microfluidic channels are in substantially the sameplane; the plurality of microfluidic channels do not intersect oneanother within the center portion; the detection window comprises a thinplastic; and the detection window overlaps the detection region of eachmicrofluidic channel.
 2. The chip of claim 1 wherein the region of thinplastic comprises polyethylene, a poly(acrylate), a poly(carbonate), anunsaturated, partially unsaturated or saturated cyclic olefin polymer(COP), an unsaturated, partially unsaturated, or saturated cyclic olefincopolymer (COC), or a norbornene thermopolymer.
 3. The chip of claim 1wherein the thin plastic comprises a ZEONOR™, ZEONEX™ or TOPAS™ polymer.4. The chip of claim 1 wherein the thin plastic has a thickness of lessthan about 300 μm.
 5. The chip of claim 1 wherein the thin plastic has athickness of less than about 500 μm.
 6. The chip of claim 1 wherein thethin plastic has a thickness of one millimeter or less.
 7. The chip ofclaim 1 wherein each microfluidic channel has a separation length offrom 2 cm to 50 cm.
 8. The chip of claim 1, wherein each of theplurality of microfluidic channels further comprises an injectionchannel.
 9. The chip of claim 1 wherein the thin plastic essentiallydoes not fluoresce light having a wavelength between 500 and 800 nm whenexcited at a wavelength between about 450 and 500 nm.
 10. The chip ofclaim 9 wherein the thin plastic is excited at a wavelength of about 488nm.
 11. The chip of claim 1, wherein a plurality of nucleic acid speciesin a nucleic acid sample generated for fragment sizing applications canbe detected with a signal to noise of greater than 3 starting with asingle copy of a nucleic acid template for PCR amplification.
 12. Thechip of claim 1, wherein a plurality of nucleic acid species in anucleic acid sample generated for DNA sequencing application can bedetected with a signal to noise of greater than 3 starting with a singlecopy of a DNA template for PCR amplification.
 13. The chip of claim 1wherein each of the plurality of microfluidic channels further comprisesa surface coating.
 14. The chip of claim 13 wherein the surface coatingis hydroxypropylmethylcellulose (HPMA), poly(ethylene oxide) (PEO),poly(vinyl alcohol) (PVA), poly(dimethyl acrylamide) (PDMA),poly(vinylpyrrolidinone), dimethylacrylamide (DMA), diethylacrylamideDEA, poly(diethylacrylamide) and mixtures thereof.
 15. The chip of claim13 wherein the each of the plurality of microfluidic channels furthercomprises a sieving matrix.
 16. The chip of claim 15 wherein the sievingmatrix comprises a linear or cross-linked poly (N,N-dialkylacrylamide),linear polyacrylamide, polydimethylacrylamide, polyvinylpyrrolydinone,or combinations thereof.
 17. The chip of claim 16 wherein the sievingmatrix comprises 1-50 wt. % polyacrylamide.
 18. The chip of claim 1further comprising a porous layer between each cathode well and eachmicrofluidic channel, wherein the porous layer is capable ofsubstantially blocking passage of gas bubbles from the cathode wellsinto each microfluidic channel.
 19. The chip of claim 18 wherein theporous layer comprises a glass frit, a polymer frit, a polymer membrane,or a polymer filter.
 20. The chip of claim 1, wherein a plurality ofnucleic acid species in a nucleic acid sample can be detected withsingle-base resolution after electrophoresis analysis of said nucleicacid sample.
 21. The chip of claim 1, further comprising an injector forsimultaneously injecting at least two samples into the plurality ofmicrofluidic channels.
 22. An apparatus comprising a support having atop and bottom surface, comprising an anode portion, a cathode portion,and a center portion between the anode and cathode portions, wherein thecenter portion comprises an aperture at the detection window, the anodeportion comprises the at least one anode well, and the cathode portioncomprises the at least one cathode well; the apparatus furthercomprising a chip according to claim 1, having a top and bottom surface,wherein the top surface of the chip is in contact with the bottomsurface of the support, the microfluidic channels are in fluidcommunication with the cathode and anode wells through the vias; and thechip is fixedly attached to the support.
 23. The apparatus of claim 22,wherein the separation chip comprises a substrate layer and a coverlayer each having a top and bottom surface, wherein the substrate layercomprises a plurality of microfluidic grooves in its top surface; andthe cover layer comprises one via for each anode and cathode well of thechip support, wherein the top surface of the substrate layer is incontact with the bottom surface of the cover layer, thereby togetherforming the plurality of microfluidic channels in the separation chip.24. The apparatus of claim 23, wherein the substrate layer and coverlayer are thermally bonded.
 25. A method for electrophoreticallyseparating and detecting a plurality of samples simultaneouslycomprising, providing a plurality of samples into each of a plurality ofmicrofluidic channels on a microchip according to claim 1; applying anelectric potential across the plurality of microfluidic channels toseparate detectable species comprising each of the plurality of analysissamples; detecting each of the detectable species comprising theplurality of separated samples at the detection window.
 26. The methodof claim 25, further comprising the step of maintaining a substantiallyidentical electric field across each of the plurality of microfluidicchannels.
 27. The method of claim 26, wherein an essentially identicalelectric field is maintained across each of the plurality ofmicrofluidic channels by balancing the resistance of each portion ofeach of the plurality of microfluidic channels.
 28. The method of claim25, wherein the detectable species comprise nucleic acids.
 29. Themethod of claim 28, wherein the detectable species comprise dyesfunctionally attached to the nucleic acids.